Saccharomyces cerevisiae culture and its application

Abstract

The present invention provides a saccharomyces cerevisiae culture and its application. The saccharomyces cerevisiae culture is obtained by utilizing food processing byproducts such as soybean molasses, pomace, and coffee grounds under specific conditions using the saccharomyces cerevisiae strain HKB-145007 (deposit number CGMCC No. 24839). The saccharomyces cerevisiae culture is rich in active substances such as protein, mannooligosaccharides, small peptides, organic acids, and polyphenols. When added to a complete mixed diet for dairy cows at a ratio of 3% to 5%, or used in combination with a drug for treating mastitis in dairy cows, it has a positive effect on preventing and treating mastitis in dairy cows.

Description

Technical Field

[0001] The invention relates to the fields of feed and prevention and treatment of dairy cow mastitis, and in particular to a brewer's yeast culture and application thereof. Background Art

[0002] Mastitis is a long-standing and critical disease plaguing dairy cow production. It not only leads to decreased milk production, increased culling rates, and mortality, but also reduces milk quality and increases feeding costs, resulting in significant annual losses to the global dairy industry. Data shows that the clinical incidence of mastitis on large-scale intensive dairy farms is 3.3%, while on family farms it is over 10%, resulting in economic losses exceeding 600 million yuan. Currently, clinical treatment for mastitis primarily relies on antibiotics, but issues such as antibiotic residues and resistance have raised concerns about biosafety and food safety, necessitating strict control of antibiotic use in dairy farming. Therefore, the development and use of novel additives to prevent and treat mastitis in dairy cows is a key issue for the healthy and sustainable development of the dairy industry.

[0003] Food processing byproducts such as soybean molasses, pomace, and coffee grounds are rich in dietary fiber, and multiple studies have shown their potential for use in ruminant farming (Han Qipeng et al., 2015; Liu Shumin et al., 2013; Shi Jinjie et al., 2022). However, these food processing byproducts contain anti-nutritional factors such as saponins and tannins, which affect palatability and digestibility. Their high sugar and moisture content also leads to rancidity and spoilage, preventing their direct use in large quantities. Furthermore, because pomace and coffee grounds are rich in sugars such as fructose, raffinose, and stachyose, which are inaccessible to animals and common fermentation strains, their silage and fermentation potential has been underdeveloped. These factors severely limit the use of these food processing byproducts in animal husbandry and result in varying degrees of resource waste and environmental pollution. In today's global food resource crunch, the rational development and utilization of food processing byproducts is a key approach to improving the quality and efficiency of my country's agriculture.

[0004] Brewer's yeast culture has been widely used in ruminant farming since the 1990s. It uses yeast as the main strain and adopts a liquid-solid fermentation model. It is produced through a fermentation process that combines aerobic liquid fermentation and anaerobic solid-state fermentation. It is composed of yeast extracellular metabolites, denatured culture medium and a small amount of inactive yeast cells. It is rich in active ingredients such as nutrients, organic acids, enzymes, aromatic substances and unknown factors.

[0005] With technological advancements, its preparation techniques and targeted applications have been gradually developed. Its primary effect is on the rumen, with several benefits, including stabilizing ruminal pH, improving ruminal fermentation, increasing fiber digestibility, increasing feed intake, and enhancing animal performance. In the current context of antibiotic prohibitions in feed and antibiotic reduction in livestock farming, the immune-modulating effects of Saccharomyces cerevisiae culture on dairy cows are gaining increasing attention. Various studies have found that it can alleviate mastitis to varying degrees (Guo Ting, 2021; Huo Yanming, 2020; Liu Dacheng, 2011). Feed premixes for mastitis prevention have also been prepared using Saccharomyces cerevisiae culture and medicinal plants such as dandelions (Duan Hongfeng, 2017; Zhang Tingzhou, 2014; Wang Hong, 2014). However, there are currently no reports on the development of functional Saccharomyces cerevisiae culture products for treating mastitis in dairy cows through fermentation in specific liquid or solid media. Summary of the Invention

[0006] The purpose of the present invention is to provide a brewer's yeast culture and application thereof.

[0007] To achieve the objectives of the present invention, in a first aspect, the present invention provides an antibacterial yeast strain, Saccharomyces cerevisiae HKB-145007, which has been deposited at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 24839 and a deposit date of May 6, 2022. This Saccharomyces cerevisiae strain can effectively utilize sugars such as sucrose, fructose, raffinose, and stachyose in culture media, and can reduce the content of substances such as saponins and tannins in food processing byproducts such as pomace and coffee grounds.

[0008] In a second aspect, the present invention provides a bacterial agent containing the Saccharomyces cerevisiae.

[0009] In a third aspect, the present invention provides a culture of brewer's yeast, which is obtained by inoculating the brewer's yeast with food processing by-products as a culture medium for fermentation.

[0010] The food processing by-products include, but are not limited to, soy molasses, pomace (such as grape pomace), coffee grounds, and wheat bran.

[0011] In a fourth aspect, the present invention provides a method for preparing a culture of Saccharomyces cerevisiae, comprising the following steps:

[0012] (1) Preparation of seed solution;

[0013] (2) Liquid aerobic fermentation;

[0014] (3) solid anaerobic fermentation;

[0015] (4) The solid fermentation product is broken to obtain the finished product of brewer's yeast culture.

[0016] Furthermore, the brewer's yeast is used as the fermentation strain, the liquid culture medium used in step (2) is diluted soybean molasses, and yeast liquid is obtained by aerobic fermentation, and the yeast content is ≥5g / L (dry weight of yeast);

[0017] Furthermore, step (3) includes: mixing wheat bran, grape pomace, and coffee grounds in a mass ratio of 2:2:1 and sterilizing at high temperature to prepare a solid fermentation raw material; mixing soybean molasses and the yeast solution of step (2) in a mass ratio of 1:2 to prepare a starter; mixing the starter and the solid fermentation raw material in a mass ratio of 1:2.5, adjusting the moisture content of the fermentation material to 45-55% (preferably 50%) with water, and performing anaerobic fermentation;

[0018] Furthermore, step (4) includes: after the anaerobic fermentation is completed, adding 1% of the yeast solution of step (2) and 1.2% of the cell wall breaking agent to the solid fermentation product by mass, mixing and sealing for 3 hours, and drying and crushing the fermentation product to prepare a finished product of brewer's yeast culture.

[0019] Preferably, the liquid culture medium used in step (2) is prepared by diluting soybean molasses with water to 2-3 times the mass of soybean molasses and adjusting the pH value to 5.7-6.5.

[0020] Furthermore, step (1) comprises: inoculating the yeast strain activated by the slant strain into a liquid seed culture medium containing 145 g / L malt extract powder and 0.08 g / L chloramphenicol, with a pH value of 6.0, and culturing in a constant temperature shaker at 28° C. for 32-35 h (preferably 32 h) at a rotation speed of 150 r / min to obtain a seed liquid.

[0021] Furthermore, the conditions for aerobic fermentation in step (2) are as follows: an inoculum amount of 22%-30% (volume percentage) of the seed solution, a fermentation temperature of 26° C.-30° C., a fermentation time of 34-40 hours, and an aeration rate of 2-3.5 L / min, to obtain a yeast solution having a yeast content of ≥5 g / L (dry weight of yeast).

[0022] Furthermore, the conditions for anaerobic fermentation in step (3) are: fermentation temperature 26-32° C., and fermentation time 30-34 h.

[0023] In a fifth aspect, the present invention provides any of the following uses of the cerevisiae yeast and / or its culture:

[0024] 1) Used for the preparation of antibacterial biological products;

[0025] 2) Used for preparing biological products for preventing and treating mastitis in dairy cows;

[0026] 3) Used as feed additive;

[0027] 4) Used for livestock and poultry breeding.

[0028] The bacteria include but are not limited to Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, and Pseudomonas aeruginosa.

[0029] The brewer's yeast culture of the present invention is rich in active substances such as protein, mannooligosaccharide, small peptide, organic acid, polyphenol and the like, and can be added to a complete mixed diet of dairy cows at a ratio of 3% to 5%, or used in combination with a drug for treating dairy cow mastitis, to have a positive improvement effect on the prevention and treatment of dairy cow mastitis.

[0030] In a sixth aspect, the present invention provides a feed comprising the following ingredients in parts by weight: 3.00-5.00 parts of brewer's yeast culture, 22 parts of whole-plant silage corn, 12-13 parts of alfalfa hay, 4.5-5.5 parts of oat grass, 3.0-3.5 parts of beet pulp, 7-7.5 parts of whole cottonseed, 21.5-22.5 parts of corn, 5.0-5.5 parts of corn DDGS, 7.0-7.5 parts of soybean meal, 3.0-3.5 parts of wheat bran, 1.0-1.5 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of stone powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine and 0.02 parts of rumen bypass tryptophan.

[0031] Preferably, the feed comprises the following ingredients in parts by weight: 3.00 parts of brewer's yeast culture, 23.54 parts of whole-plant corn silage, 13.52 parts of alfalfa hay, 5.55 parts of oat grass, 3.50 parts of beet pulp, 7.50 parts of whole cottonseed, 22.50 parts of corn, 5.50 parts of corn DDGS, 7.50 parts of soybean meal, 3.60 parts of wheat bran, 1.50 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of stone powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine and 0.02 parts of rumen bypass tryptophan.

[0032] Among them, each kilogram of vitamin and mineral premix contains: vitamin A 600,000 IU, vitamin D 80,000 IU, vitamin E 5,000 IU, iron 3,000 mg, copper 2,000 mg, manganese 2,500 mg, zinc 8,000 mg, iodine 100 mg, selenium 60 mg and cobalt 20 mg.

[0033] In a seventh aspect, the present invention provides use of the feed in breeding ruminants (such as cattle, preferably dairy cows).

[0034] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] (1) The brewer's yeast culture of the present invention is produced by fermenting the brewer's yeast HKB-145007 by making full use of food processing by-products such as soybean molasses and pomace through a specific process, which has a promoting effect on improving the quality and efficiency of my country's agriculture.

[0036] (2) The method for preparing a brewer's yeast culture provided by the present invention fully utilizes sucrose, fructose, and sugars such as raffinose and perilla sugar in the culture medium that cannot be fermented and utilized by general yeast, thereby improving the utilization rate of the sugar source; and reducing anti-nutritional factors such as saponins and tannins in the fermentation substrate that affect palatability and nutrient digestion and absorption.

[0037] (3) The brewer's yeast culture of the present invention is rich in small peptides, mannans and other yeast metabolites, and has a high immune regulating effect on dairy cows.

[0038] (4) The brewer's yeast culture of the present invention is added to the TMR diet of dairy cows, with 50-200 g / head / day added according to the different physiological needs of dairy cows, which can effectively prevent and control mastitis in dairy cows and ensure the milk production, milk quality and other lactation performance of dairy cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The figure shows the effect of Saccharomyces cerevisiae culture (YC) on MACT cell activity in a preferred embodiment of the present invention. Wherein, * indicates that the difference between different treatment groups is statistically significant, P<0.05.

[0040] Figure 2 The protective effect of Saccharomyces cerevisiae culture (YC) on LPS-challenged MACT cells (100 μm) in a preferred embodiment of the present invention is shown.

[0041] Figure 3 The figure shows the effect of Saccharomyces cerevisiae culture (YC) on oxidative stress of LPS-challenged MACT cells in a preferred embodiment of the present invention.

[0042] Figure 4Figure 2 shows the effect of Saccharomyces cerevisiae culture on the secretion of inflammatory factors by LPS-challenged MACT cells in a preferred embodiment of the present invention. Different lowercase letters indicate statistically significant differences between different treatment groups, P < 0.05.

[0043] Figure 5 The effect of YC on milk somatic cells of cows with subclinical mastitis in a preferred embodiment of the present invention is shown in FIG. Different lowercase letters indicate that the differences between different treatment groups are statistically significant, with P < 0.05.

[0044] Figure 6 The effect of YC on milk production of cows with subclinical mastitis in a preferred embodiment of the present invention is shown in FIG. Different lowercase letters indicate that the differences between different treatment groups are statistically significant, P<0.05.

[0045] Figure 7 This is the auxiliary effect of YC in treating mastitis in dairy cows in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0046] The present invention provides a brewer's yeast culture prepared using food processing by-products such as soybean molasses and pomace as a culture medium. The product can be used for the prevention and treatment of mastitis in dairy cow breeding, so as to ensure milk production and milk quality in dairy cow breeding and improve the lactation performance of dairy cows.

[0047] According to a first aspect of the present invention, there is provided a culture of Saccharomyces cerevisiae prepared using food processing by-products such as soybean molasses and pomace as a culture medium.

[0048] The brewer's yeast culture is obtained by fully fermenting food processing by-products such as soybean molasses and pomace by the brewer's yeast HKB-145007 under specific conditions.

[0049] The brewer's yeast culture is prepared by the following steps: activating the brewer's yeast HKB-145007 by slant culture and then inoculating it into seed liquid for propagation; inoculating the propagated seed liquid into soybean molasses for liquid aerobic fermentation; then inoculating it into a solid culture medium containing soybean molasses for solid anaerobic fermentation; and drying and pulverizing the obtained solid fermentation product.

[0050] Among them, in the seed liquid obtaining step, the slant strain is activated: the slant strain is activated on the solid malt wort culture medium, the culture temperature is 28°C, and the culture time is 38h; then the seed liquid is obtained by fermentation and expansion on the liquid seed culture medium, the culture temperature is 28°C, the rotation speed is 150r / min; the culture time is 32h.

[0051] In the liquid aerobic fermentation step, the liquid culture medium is diluted soybean molasses, water is added to dilute the soybean molasses to 2-3 times the mass of the soybean molasses, and the pH value is adjusted to 5.7-6.5.

[0052] In the liquid aerobic fermentation step, the inoculum amount of the seed liquid is 22%-30%, the fermentation temperature is 26°C-30°C, the fermentation time is 34-40 hours, and the ventilation volume is 2-3.5 L / min to obtain a yeast solution with a yeast content of ≥5 g / L (dry weight of yeast).

[0053] Among them, the solid anaerobic fermentation step includes: solid fermentation raw materials such as wheat bran, grape pomace, and coffee grounds, with a mass ratio of wheat bran, grape pomace, and coffee grounds of 2:2:1, mixed and sterilized at high temperature; 1 part of soy molasses and 2 parts of yeast liquid are fully mixed to obtain a starter; the starter and the solid fermentation raw materials are mixed at a mass ratio of 1:2.5, and water is added to adjust the moisture content of the material before fermentation to 50%.

[0054] Wherein, in the solid anaerobic fermentation step, the fermentation temperature is 26-32° C. and the fermentation time is 30-34 hours.

[0055] Among them, the brewer's yeast culture finished product step includes adding 1% yeast liquid and 1.2% wall-breaking agent after the fermentation is completed, mixing and sealing for 3 hours, drying and crushing the fermentation product to make the brewer's yeast culture finished product.

[0056] According to a second aspect of the present invention, there is provided a use of the cerevisiae yeast culture in preventing and treating mastitis in dairy cows.

[0057] Among them, this product is mixed and fed in the TMR diet of dairy cows. The recommended diet formula is: 3.00 parts of brewer's yeast culture, 23.54 parts of whole corn silage, 13.52 parts of alfalfa hay, 5.55 parts of oat grass, 3.50 parts of beet pulp, 7.50 parts of whole cottonseed, 22.50 parts of corn, 5.50 parts of corn DDGS, 7.50 parts of soybean meal, 3.60 parts of wheat bran, 1.50 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of stone powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine, and 0.02 parts of rumen bypass tryptophan.

[0058] Among them, each kilogram of vitamin and mineral premix contains: vitamin A 600,000 IU, vitamin D 80,000 IU, vitamin E 5,000 IU, iron 3,000 mg, copper 2,000 mg, manganese 2,500 mg, zinc 8,000 mg, iodine 100 mg, selenium 60 mg, and cobalt 20 mg.

[0059] According to a third aspect of the present invention, there is provided an application of the brewer's yeast culture product for preventing and controlling mastitis in dairy cows and ensuring milk production and quality.

[0060] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0061] The main components of the wall-breaking agent used in the following examples are β-glucanase, mannanase, acid protease, cellulase and zinc sulfate mixture. The above enzyme preparations and zinc sulfate were purchased from Wuhan Yuancheng Gongchuang Technology Co., Ltd.

[0062] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0063] Example 1 Isolation of Saccharomyces cerevisiae

[0064] (1) Culture medium: malt extract culture medium. Take 500g of malt, crush it and put it into a beaker, add 2000mL of distilled water, stir and place in a 45℃ water bath for 30min, then raise the temperature to 70℃ and maintain for 1h; filter through 8 layers of gauze and wash the beaker and filter trough with shampoo until the filtrate reaches 2000mL. Bring to a boil, cool and store at 10℃ for later use.

[0065] (2) Yeast isolation: Beer, wine, pickles, fermented fruit and vegetable juice, dough, and kumis collected from Xinjiang Uygur Autonomous Region, Inner Mongolia Autonomous Region, and Shandong Province were used as raw materials. 400 mL (liquid sample) or 400 g (crushed solid sample) of raw materials were collected and placed in a sterile conical flask, covered with 8 layers of gauze, and naturally fermented at 28°C for 3 days. Under sterile conditions, the fermentation culture sample was shaken evenly, and an appropriate amount of fermentation liquid was pipetted into sterilized malt wort culture medium with a sterile pipette and incubated at 28°C. When the wine smell was present, the culture was diluted to a concentration of 10-4 using a gradient dilution method. 0.1 mL of the diluted culture was evenly spread on a malt wort culture medium (malt wort culture medium + 2% agar), and inverted at 28°C for 48 hours. After colonies grew, typical single colonies (yeast colonies) were selected and further streaked and purified three times. After microscopic examination showed that the culture was pure, the culture was transferred to a solid slant medium of malt wort and stored at 4°C for later use.

[0066] The yeasts were cultured in malt extract medium and their growth was observed over a period of time. The strains with the highest survival rate and most vigorous growth were selected for the next step of degradation of soy molasses oligosaccharides. A total of nine yeast strains were obtained: HKB-145001, HKB-145002, HKB-145003, HKB-145005, HKB-145006, HKB-145007, HKB-145008, and HKB-145009.

[0067] Example 2 Screening of Saccharomyces cerevisiae

[0068] (1) Soybean molasses fermentation: Under sterile conditions, two loops of inoculating loops were taken from the malt wort solid slant culture medium of the eight strains of Saccharomyces cerevisiae screened in Example 1, which had been kept aside. The loops were then inoculated into soybean molasses (without other ingredients). The pH of the soybean molasses culture medium was adjusted to 6.2, and the fermentation temperature was set at 28°C for 36 h. A blank control group without yeast inoculation was also set up.

[0069] (2) The contents of raffinose, stachyose, and saponin in unfermented soybean molasses and soybean molasses fermented with yeast were determined. The results are shown in Table 1.

[0070] Table 1 Effects of different yeast fermentations on the contents of raffinose, stachyose and saponin in soybean molasses

[0071]

[0072]

[0073] As shown in Table 1, soybean molasses contains high levels of raffinose, stachyose, and saponins. When fermentation alone was used without yeast inoculation, the raffinose, stachyose, and saponin contents remained unchanged (blank control group). Among the nine strains of Saccharomyces cerevisiae, HKB-145007 showed the best degradation of raffinose, stachyose, and saponins in soybean molasses, with degradation rates reaching 81.18%, 79.96%, and 74.55%, respectively.

[0074] Example 3 Preparation of Saccharomyces cerevisiae culture by fermentation of soy molasses, pomace and other by-products

[0075] (1) Slant culture activation: The frozen stored Saccharomyces cerevisiae HKB-145007 was inoculated onto a slant culture medium containing 130 g / L malt extract powder, 0.1 g / L chloramphenicol, and 20 g / L agar, and cultured at a constant temperature of 28°C for 38 h to obtain the activated yeast culture.

[0076] (2) Preparation of seed liquid: One loop of activated yeast strain was inoculated into a liquid culture medium containing 145 g / L malt extract powder, 0.08 g / L chloramphenicol, and a pH value of 6.0. The culture was incubated in a constant temperature shaker at 28°C and a rotation speed of 150 r / min for 32 h.

[0077] (3) Soybean molasses fermentation (liquid aerobic fermentation): The prepared seed liquid was inoculated into the diluted soybean molasses culture medium at a 25% inoculum volume. The dilution ratio of soybean molasses was 2.5 times, that is, water was added to dilute the soybean molasses to 2.5 times the mass of soybean molasses. The pH value was 6.0, and the culture was expanded. The ventilation volume was controlled at 3L / min, the temperature was 28℃, and the fermentation time was 32h. Yeast culture liquid was obtained. The yeast content was ≥5g / L (dry weight of yeast). (4) Solid anaerobic fermentation: solid fermentation raw materials such as wheat bran, grape pomace and coffee grounds were mixed, sterilized and cooled, and then mixed with 1 part of coffee grounds to obtain solid fermentation raw materials; 1 part of soybean molasses and 2 parts of yeast culture liquid were fully mixed to obtain a fermentation agent; the fermentation agent and solid fermentation raw materials were mixed at a mass ratio of 1:2.5, and the moisture content of the material before fermentation was adjusted to 50%; anaerobic fermentation was carried out in a temperature-controlled fermentation tank at a fermentation temperature of 28℃ and a fermentation time of 30h. Obtain solid-state fermentation products.

[0078] (5) Obtaining a brewer's yeast culture product: Adding 1% yeast solution (yeast culture solution) of the fermentation product by mass and 1.2% cell wall breaking agent to the solid fermentation product, stirring and mixing, and after the cell wall breaking agent acts for 3 hours, drying and crushing are performed to obtain a brewer's yeast culture product.

[0079] (6) The contents of the main components in the Saccharomyces cerevisiae culture were determined, as shown in Table 2. The prepared Saccharomyces cerevisiae culture was rich in protein, mannan, organic acid and polyphenols, and the anti-nutritional factors such as saponin and tannin were reduced by more than 60%.

[0080] Table 2 Main components of the culture of Saccharomyces cerevisiae prepared by fermenting soy molasses (dry matter basis, %)

[0081]

[0082] Example 4 Inflammatory Effects of Saccharomyces cerevisiae Culture on Cow Mammary Epithelial Cells (MACT Cells)

[0083] (1) Establishment of a bovine mammary epithelial cell (MACT cell) model: MACT cells were cultured in DMEM containing 10% FBS and 200 U / mL streptomycin / penicillin at 37°C in an incubator containing 5% carbon dioxide. The culture medium was replaced every 48 h, and the cells were lysed at 80% to 90% confluency using 0.25% trypsin solution; MACT cells were then detached using 0.25% trypsin-ethylenediaminetetraacetic acid.

[0084] (2) Preparation of Saccharomyces cerevisiae culture (YC) suspension: Weigh 10g of YC and put it into 40mL of ultrapure water solution, vortex it to mix it thoroughly and incubate it on a shaker at room temperature for 1h to obtain a YC suspension with a concentration of 20.0%. Then dilute it with ultrapure water to prepare 1.0%, 5.0% and 10.0% YC suspensions. (3) Detection of the toxicity of YC on MACT cells: Using the MTT method, cells were inoculated in 96-well plates, and 0.0% (ultrapure water), 1.0%, 5.0%, 10.0% and 20.0% YC suspensions were added dropwise. After standing for 24h, MTT (10μL / 0.5mg / mL) was added to the 96-well plates, and acidic isopropanol was added 4h later. The optical density at 550nm was measured using a microplate reader and used to calculate the cell survival rate. The results showed that only high concentrations above 20% would have adverse effects on bovine mammary cells ( Figure 1 ).

[0085] (4) Lipopolysaccharide (LPS) challenge: MACT cells were isolated using 0.25% trypsin-ethylenediaminetetraacetic acid, and 0.0%, 1.0%, 5.0%, and 10.0% YC were added to the cells. One hour later, MACT cells were stimulated with 1 μg / mL LPS for 6 hours.

[0086] (5) Electron microscopic observation of MACT cells at 100 μm magnification revealed that LPS could significantly damage cell activity, while 1.0% YC could only slightly protect MACT cells from damage. However, 5.0% and 10.0% YC could effectively protect MACT cells from cell loss caused by LPS attack ( Figure 2 ).

[0087] (6) The fluorescence microplate reader method was used to detect the effect of YC on the oxidative stress relief of MACT cells after LPS challenge. By comparing the oxidative stress markers reactive oxygen species (ROS) and glutathione (GSH), it was found that YC at a concentration of 5.0% or more could significantly relieve the oxidative stress of MACT cells after LPS co-exposure, and the effect was dose-dependent ( Figure 3 ).

[0088] (7) Enzyme-linked immunosorbent assay to determine inflammatory factors: ELISA kits were used to detect the levels of TNF-α, IL-6, and IL-1β (pg / ml). The results showed that LPS challenge significantly promoted the secretion of inflammatory factors, while the addition of YC effectively alleviated the inflammatory response of cells ( Figure 4 ).

[0089] In summary, cow mammary epithelial cells (MACT cells) are the direct cell tissue of the cow's mammary inflammatory response. In this embodiment, LPS-induced toxicity simulation of mammary cells showed cellular characteristics of mammary inflammation under toxin stimulation, and cell oxidative stress and inflammatory factor secretion were aggravated. In the experimental group pretreated with YC, the oxidative stress presented by MACT cells was reduced, and proinflammatory cytokines were suppressed, indicating that cell tissue is actively fighting inflammatory pathology, preventing or stopping the progression of bovine mastitis. This provides basic theory and data support for YC for the prevention and control of cow mastitis.

[0090] Example 5 Study on the antibacterial effect of Saccharomyces cerevisiae culture in vitro

[0091] The antibacterial effects of Saccharomyces cerevisiae culture on the main pathogens of cow mastitis, Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa and pathogenic Escherichia coli, were observed and compared using the Oxford cup method.

[0092] (1) Experimental instruments

[0093] Clean bench, high-pressure steam sterilizer, centrifuge, constant temperature incubator, constant temperature blast drying oven, water bath thermostat, electronic balance, pH meter, constant temperature shaker, petri dish (90mm diameter), pipette, inoculation loop, Oxford cup, vernier caliper

[0094] (2) Experimental materials

[0095] Test samples: Saccharomyces cerevisiae culture A (HKB-145007), antibacterial Saccharomyces cerevisiae culture B ( A100, HKB-36 fermentation preparation), Saccharomyces cerevisiae culture C ( Universal type).

[0096] Indicator bacteria: Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa and pathogenic Escherichia coli.

[0097] LB solid medium: 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L peptone. Dissolve the mixture and adjust the pH to 7.2-7.4 with NaOH solution. Add 1.5% agar, bring to a boil, divide into flasks, autoclave at 121°C for 15 min, and place in a 50-55°C water bath until ready for use.

[0098] LB liquid medium: 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L peptone. After dissolving, adjust the pH to 7.2-7.4, divide the medium into flasks, and sterilize it by autoclaving at 121°C for 15 min.

[0099] Normal saline: Dissolve 8.5 g of sodium chloride in 1000 mL of distilled water and sterilize by autoclaving at 121°C for 15 min.

[0100] (3) Solution preparation

[0101] Indicator bacteria suspension: Streak the indicator bacteria onto an LB solid plate and incubate at 37°C for 12 h. Pick a single colony and transfer it to LB liquid medium (5 mL / tube). Incubate it in a shaker at 37°C and 150 rpm for 24 h. Store in a refrigerator at 4°C until use.

[0102] Test solution: Accurately weigh 2 g of yeast culture sample and dissolve it in 6 mL of sterile saline (1:3 dissolution). Shake and dissolve for 30 min. Centrifuge at 4000 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm microporous membrane for later use. This is the test solution.

[0103] (4) Preparation of antibacterial plates (tube plate method): Add the indicator bacteria suspension to LB solid medium cooled to 50°C. Add 20 μL of the indicator bacteria solution to 100 mL of the medium, shake well, and transfer 15 mL to a plate. Place the plate in a horizontal laminar flow hood and wait for it to completely solidify. To ensure complete diffusion of the test solution, the newly prepared antibacterial plate should be placed in a 4°C refrigerator for 24 hours before use.

[0104] (5) Determination of antibacterial activity

[0105] Place sterilized Oxford cups evenly on the antibacterial plates and add 100 μL of the test solution to each Oxford cup. Carefully move the plates and place them in a 4°C refrigerator in the forward direction for 6–12 hours to allow for diffusion. Place the infiltrated plates in a constant-temperature incubator at 37°C for 16–18 hours.

[0106] Make three parallel incubations for the test solution. After the incubation is complete, measure the diameter of the inhibition zone with a vernier caliper.

[0107] The antibacterial effects in Table 3 show that the saccharomyces cerevisiae culture A of the present invention has the best antibacterial effect on Staphylococcus aureus, followed by Streptococcus agalactiae, and is better than the antibacterial saccharomyces cerevisiae culture B in antibacterial effect on Streptococcus agalactiae and Pseudomonas aeruginosa, two major mastitis pathogens. The overall antibacterial effect is better than that of the ordinary saccharomyces cerevisiae culture C.

[0108] Table 3 Antibacterial effect of Saccharomyces cerevisiae culture in vitro (diameter of inhibition zone / mm)

[0109] indicator bacteria Staphylococcus aureus Streptococcus agalactiae Pseudomonas aeruginosa Escherichia coli Yeast culture A 20.5 21.9 18.1 17.8 Yeast culture B 18.1 14.2 15.1 18.0 Yeast culture C 14.3 12.1 13.4 12.1

[0110] The antibacterial effect of Saccharomyces cerevisiae culture on a mixed indicator bacteria (equal parts of Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa, and pathogenic Escherichia coli) was tested at different pH values. The results in Table 4 show that the Saccharomyces cerevisiae culture (A) of the present invention can maintain antibacterial activity in the rumen and has a superior inhibitory effect on the mixed indicator bacteria compared to the antibacterial Saccharomyces cerevisiae culture (B) and the conventional Saccharomyces cerevisiae culture (C).

[0111] Table 4 Antibacterial effect of Saccharomyces cerevisiae culture on mixed bacteria under different pH treatments (inhibition zone diameter / mm)

[0112] Treatment pH and time 5.5，60s 6.5，60s 7，60s 7.5，60s Yeast culture A 19.5 19.5 19.3 19.4 Yeast culture B 17.2 17.2 17.3 17.3 Yeast culture C 13.5 13.4 13.5 13.4

[0113] Example 6 Application of Saccharomyces cerevisiae culture in improving latent mastitis in dairy cows

[0114] 1 Materials and Methods

[0115] 1.1 Experimental Design

[0116] Thirty Holstein-Friesian dairy cows (2-4 parities, 60-80 days of lactation, with a lactating somatic cell count greater than 300,000 units) were randomly divided into a control group, experimental group 1, and experimental group 2, with 10 cows in each group and each cow as a replicate. The experimental period was 30 days, with a 2-day pre-feeding period and a 28-day experimental period.

[0117] 1.2 Diet composition

[0118] The diet of the control group included 25.54 parts of whole corn silage, 14.52 parts of alfalfa hay, 5.55 parts of oat hay, 3.50 parts of beet pulp, 7.50 parts of whole cottonseed, 22.50 parts of corn, 5.50 parts of corn DDGS, 7.50 parts of soybean meal, 3.60 parts of wheat bran, 1.50 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of stone powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of table salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine, and 0.02 parts of rumen bypass tryptophan.

[0119] The diet of Experiment 1 group was as follows: 3.00 parts of the brewer's yeast culture obtained in Example 1, 23.54 parts of whole-plant corn silage, 13.52 parts of alfalfa hay, 5.55 parts of oat straw, 3.50 parts of beet pulp, 7.50 parts of whole cottonseed, 22.50 parts of corn, 5.50 parts of corn DDGS, 7.50 parts of soybean meal, 3.60 parts of wheat bran, 1.50 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of rock powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine, and 0.02 parts of rumen bypass tryptophan.

[0120] Among them, each kilogram of vitamin and mineral premix contains: vitamin A 600,000 IU, vitamin D 80,000 IU, vitamin E 5,000 IU, iron 3,000 mg, copper 2,000 mg, manganese 2,500 mg, zinc 8,000 mg, iodine 100 mg, selenium 60 mg, and cobalt 20 mg.

[0121] Experimental 2 diet: common cerevisiae culture ( General type) 3.00 parts, whole plant silage corn 23.54 parts, alfalfa hay 13.52 parts, oat straw 5.55 parts, beet pulp 3.50 parts, whole cottonseed 7.50 parts, corn 22.50 parts, corn DDGS 5.50 parts, soybean meal 7.50 parts, wheat bran 3.60 parts, cottonseed meal 1.50 parts, baking soda 0.60 parts, stone powder 0.60 parts, calcium hydrogen phosphate 0.50 parts, salt 0.30 parts, magnesium oxide 0.12 parts, vitamin and mineral premix 0.50 parts, rumen bypass lysine 0.08 parts, rumen bypass methionine 0.05 parts, rumen bypass threonine 0.02 parts, rumen bypass tryptophan 0.02 parts.

[0122] Among them, each kilogram of vitamin and mineral premix contains: vitamin A 600,000 IU, vitamin D 80,000 IU, vitamin E 5,000 IU, iron 3,000 mg, copper 2,000 mg, manganese 2,500 mg, zinc 8,000 mg, iodine 100 mg, selenium 60 mg, and cobalt 20 mg.

[0123] 1.3 Feeding and management

[0124] All experimental cows were housed in the same barn, with one pen each for the experimental and control groups. During the experimental period, the experimental cows were fed and watered freely according to the regulations of the experimental farm, and were mechanically milked.

[0125] 1.4 Measurement indicators

[0126] The somatic cell count in milk and milk yield of each cow were monitored on days 0, 7, 14, and 28 of the experiment.

[0127] 2 Results

[0128] The somatic cell count (SCC) in milk is a direct indicator of mastitis and is directly related to the health of the cow's udder and the quality of milk. It is generally believed that cows with milk containing 200,000 to 400,000 somatic cells have latent mastitis. After the brewer's yeast culture of the present invention is fed to latent mastitis cows for 28 days, the somatic cell count ( Figure 5 ) The results showed that the brewer's yeast culture of the present invention can reduce the milk somatic cell count to below 200,000 units within 2 weeks and maintain it at a healthy level, and the milk production results ( Figure 6 ) showed the same trend. It can be seen that the brewer's yeast culture of the present invention can improve the symptoms of mastitis in cows with latent mastitis within 2 weeks.

[0129] By comparing the test data of the brewer's yeast culture of the present invention (test group 1) and the general brewer's yeast culture (test group 2), it was verified that the brewer's yeast culture can improve the problem of mastitis in lactating cows. However, the somatic cell count of the general brewer's yeast culture is maintained at about 220,000 units. The purchase price of this kind of cow's milk will be significantly lower than that of cow's milk with a somatic cell count below 200,000 units.

[0130] Example 7 Application of Saccharomyces cerevisiae culture in the treatment of mastitis in dairy cows

[0131] 1 Experimental design

[0132] Four dairy cows with mastitis were selected and divided into a control group and an experimental group.

[0133] Treatment method: Mix 8 million IU penicillin and 300 mL of 10% calcium gluconate and inject it intramuscularly into the sick cows twice a day for 3 consecutive days.

[0134] Treatment in the experimental group: 8 million IU of penicillin mixed with 300 mL of 10% calcium gluconate was injected intramuscularly twice daily for 3 consecutive days. Cows were also fed 200 g of a culture of Saccharomyces cerevisiae for 10 consecutive days.

[0135] 2 Detection indicators

[0136] The number of milk somatic cells was detected on days 0, 3, 7 and 10.

[0137] 3 Results

[0138] For dairy cows confirmed to have mastitis if their somatic cell count is greater than 400,000 units, drug treatment is usually used. Only when the somatic cell count of the milk is below 200,000 units after taking the medicine and taking a one-week drug-free period can it be used for dairy product production and processing. Figure 7 The results showed that the brewer's yeast culture of the present invention, when used in combination with drug treatment, can significantly promote the therapeutic effect, and the somatic cell count of the diseased cows is within the normal range after 10 days, which can effectively reduce the breeding losses caused by mastitis in dairy cow breeding.

[0139] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Saccharomyces cerevisiae Saccharomyces cerevisiae HKB-145007, characterized in that The deposit number is CGMCC No. 24839.

2. A bacterial agent containing the brewer's yeast according to claim 1.

3. A culture of Saccharomyces cerevisiae, characterized in that The method for preparing the saccharomyces cerevisiae culture comprises the following steps: (1) Preparation of seed solution; (2) Liquid aerobic fermentation; (3) Solid anaerobic fermentation; (4) The solid fermentation product is broken into a culture product of Saccharomyces cerevisiae; The brewer's yeast of claim 1 is used as the fermentation strain, the liquid culture medium used in step (2) is diluted soybean molasses, and yeast liquid is obtained by aerobic fermentation, and the dry weight of the yeast is ≥5g / L; Step (3) comprises: mixing wheat bran, grape pomace and coffee grounds in a mass ratio of 2:2:1 and sterilizing at high temperature to prepare a solid fermentation raw material; mixing soybean molasses and the yeast solution of step (2) in a mass ratio of 1:2 to prepare a starter; mixing the starter and the solid fermentation raw material in a mass ratio of 1:2.5, adjusting the moisture content of the fermentation material to 45-55% with water, and performing anaerobic fermentation; Step (4) comprises: after the anaerobic fermentation is completed, adding 1% by mass of the yeast solution of step (2) and 1.2% of a cell wall-breaking agent to the solid fermentation product, mixing and sealing for 3 hours, drying and crushing the fermentation product to prepare a finished product of the brewer's yeast culture, wherein: The conditions for aerobic fermentation in step (2) are as follows: the inoculation amount of the seed liquid is 22%-30%, the fermentation temperature is 26°C-30°C, the fermentation time is 34-40h, and the ventilation volume is 2-3.5 L / min to obtain yeast liquid; The conditions for anaerobic fermentation in step (3) are: fermentation temperature 26-32°C, fermentation time 30-34h.

4. The saccharomyces cerevisiae culture according to claim 3, characterized in that The liquid culture medium used in step (2) is prepared by diluting soybean molasses with water to 2-3 times the mass of the soybean molasses and adjusting the pH value to 5.7-6.

5.

5. The saccharomyces cerevisiae culture according to claim 3 or 4, characterized in that Step (1) comprises: inoculating the yeast strain activated by the slant strain into a liquid seed culture medium containing 145 g / L malt extract powder and 0.08 g / L chloramphenicol with a pH value of 6.0, and culturing in a constant temperature shaker at 28°C for 32-35 h at a rotation speed of 150 r / min to obtain a seed liquid.

6. Any of the following uses of the Saccharomyces cerevisiae culture according to claim 3: 1) Used for the preparation of antibacterial biological products; 2) Used for the preparation of biological products for preventing and treating mastitis in dairy cows; 3) Used as feed additive; 4) Used for livestock and poultry breeding; The application is non-disease treatment.

7. The use according to claim 6, characterized in that The bacteria include Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Streptococcus agalactiae ( Streptococcus agalactiae ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

8. Feed, characterized in that The invention comprises the following ingredients in parts by weight: 3.00-5.00 parts of the brewer's yeast culture according to claim 3, 22 parts of whole corn silage, 12-13 parts of alfalfa hay, 4.5-5.5 parts of oat straw, 3.0-3.5 parts of beet pulp, 7-7.5 parts of whole cottonseed, 21.5-22.5 parts of corn, 5.0-5.5 parts of corn DDGS, 7.0-7.5 parts of soybean meal, 3.0-3.5 parts of wheat bran, 1.0-1.5 parts of cottonseed meal, 0.60 parts of baking soda, 0.60 parts of stone powder, 0.50 parts of calcium hydrogen phosphate, 0.30 parts of salt, 0.12 parts of magnesium oxide, 0.50 parts of vitamin and mineral premix, 0.08 parts of rumen bypass lysine, 0.05 parts of rumen bypass methionine, 0.02 parts of rumen bypass threonine and 0.02 parts of rumen bypass tryptophan; Among them, each kilogram of vitamin and mineral premix contains: vitamin A 600,000 IU, vitamin D 80,000 IU, vitamin E 5,000 IU, iron 3,000 mg, copper 2,000 mg, manganese 2,500 mg, zinc 8,000 mg, iodine 100 mg, selenium 60 mg and cobalt 20 mg.

9. Use of the feed according to claim 8 in ruminant breeding, characterized in that: The application is non-disease treatment.

10. The use according to claim 9, characterized in that The ruminant is a cattle.

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